Figure 1.
Effect of ponatinib on BCR-ABL-expressing cells.
(A) K562 cells were cultured at a concentration of 8×104/mL in the presence of varying concentrations of ponatinib for 72 h. The number of viable cells was calculated. Results are representative of three separate experiments. *P<0.05, ponatinib treatment versus control. (B) K562 cells were treated with ponatinib for 24 h, and total extracts were examined by immunoblot analysis with anti-phospho ABL, anti-Crk-L, anti-cleaved caspase 3, anti-PARP, and anti-β-tubulin antibodies. (C) Ba/F3 T315I cells were cultured in the presence of varying concentrations of ponatinib for 72 h. The number of viable cells was calculated. Results are representative of three separate experiments. *P<0.05, ponatinib treatment versus control. (D) Ba/F3 T315I cells were treated with ponatinib for 24 h, and total extracts were examined by immunoblot analysis with anti-phospho ABL, anti-Crk-L, anti-cleaved caspase 3, anti-PARP, and anti-tubulin antibodies.
Figure 2.
Ponatinib and vorinostat increase cell growth inhibition and induce apoptosis in BCR-ABL-expressing cells.
(A) Cells were cultured at a concentration of 8×104/mL in the presence or absence of ponatinib and/or vorinostat for 72 h. The percentage of proliferating cells was evaluated, as described in Materials and Methods. (B) K562 or Ba/F3 T315I cells were treated with ponatinib and/or vorinostat for 24 h, and total extracts were examined by immunoblot analysis with anti-phospho ABL, Crk-L, γH2A.X, cleaved caspase 3, PARP, acetyl histone H4, and tubulin antibodies. (C) Primary samples were cultured in the presence or absence of ponatinib and/or vorinostat. The number of living cells was calculated. *P<0.05 or **P<0.01, ponatinib and vorinostat treatment versus ponatinib treatment. (D) Primary cells were treated with ponatinib and/or vorinostat at the indicated concentrations for 24 h. The cells were examined by immunoblot analysis.
Figure 3.
Effects of ponatinib and vorinostat on Ba/F3 T315I cells in a xenograft model.
(A) In vivo studies were performed as described in Materials and Methods. At various time points, tumor sizes were reported. Average tumor weight per mouse was calculated and used to calculate the group mean tumor weight ± s.d (n = 5). (B) Immunohistochemical staining for hematoxylin and eosin (HE) at a magnification of 40×, Ki67 (magnification, 400×), and TUNEL (magnification, 400×) in Ba/F3 T315I xenograft sections. (C) Tumor cells treated with or without ponatinib and vorinostat were examined by immunoblot analysis.
Figure 4.
Analysis of ponatinib-resistant Ba/F3 BCR-ABL cells.
(A) Ba/F3 ponatinib-resistant cells were treated with various concentrations of imatinib, nilotinib, or dasatinib, and cellular growth was analyzed. These experiments were performed in triplicate. (B) Ba/F3 ponatinib-resistant cells were treated with imatinib, nilotinib, or dasatinib for 24 h. Whole cell lysates were analyzed by immunoblotting with phospho-specific Abl and Crk-L Abs. β-tubulin was used as the loading control.
Figure 5.
Effects of ponatinib and vorinostat on ABL TKI-resistant cells.
(A) Ba/F3 AP-R cells were cultured in the presence or absence of ponatinib and/or vorinostat and the viable cells were enumerated. (B) Ba/F3 AP-R cells were treated with the indicated concentrations of ponatinib and/or vorinostat for 24 h. Total cell extracts were analyzed by immunoblotting with phospho-specific Abl, anti-Crk-L, and anti-cleaved PARP antibodies. β-Actin was used as loading control. The results in A and B are representative of at least three reproducible experiments. (C) Ponatinib-resistant primary samples were cultured in the presence or absence of ponatinib and/or vorinostat and the viable cells were enumerated. *P<0.05 or **P<0.01, ponatinib and vorinostat treatment versus ponatinib treatment.